ABSTRACT
For nearly 25 y, the Committee on Science, Technology, and Law (CSTL), of the National Academies of Sciences, Engineering, and Medicine, has brought together distinguished members of the science and law communities to stimulate discussions that would lead to a better understanding of the role of science in legal decisions and government policies and to a better understanding of the legal and regulatory frameworks that govern the conduct of science. Under the leadership of recent CSTL co-chairs David Baltimore and David Tatel, and CSTL director Anne-Marie Mazza, the committee has overseen many interdisciplinary discussions and workshops, such as the international summits on human genome editing and the science of implicit bias, and has delivered advisory consensus reports focusing on topics of broad societal importance, such as dual use research in the life sciences, voting systems, and advances in neural science research using organoids and chimeras. One of the most influential CSTL activities concerns the use of forensic evidence by law enforcement and the courts, with emphasis on the scientific validity of forensic methods and the role of forensic testimony in bringing about justice. As coeditors of this Special Feature, CSTL alumni Tom Albright and Jennifer Mnookin have recruited articles at the intersection of science and law that reveal an emerging scientific revolution of forensic practice, which we hope will engage a broad community of scientists, legal scholars, and members of the public with interest in science-based legal policy and justice reform.
Subject(s)
Biological Science Disciplines , Forensic Medicine , Humans , Law Enforcement , Policy , Social Justice , Forensic SciencesABSTRACT
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Subject(s)
Cell- and Tissue-Based Therapy , Gene Editing , Neoplasms , Precision Medicine , Receptors, Antigen, T-Cell , T-Lymphocytes , Transgenes , Humans , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Biopsy , Cell- and Tissue-Based Therapy/adverse effects , Cell- and Tissue-Based Therapy/methods , Cytokine Release Syndrome/complications , Disease Progression , Encephalitis/complications , Gene Knock-In Techniques , Gene Knockout Techniques , Genes, T-Cell Receptor alpha , Genes, T-Cell Receptor beta , Mutation , Neoplasms/complications , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/therapy , Patient Safety , Precision Medicine/adverse effects , Precision Medicine/methods , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transgenes/genetics , HLA Antigens/immunology , CRISPR-Cas SystemsABSTRACT
Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml-1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.
Subject(s)
HIV Infections , HIV-1 , Adult , Antibodies, Neutralizing , Broadly Neutralizing Antibodies , Dependovirus/genetics , HIV Antibodies , HIV Infections/drug therapy , HumansABSTRACT
The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge for understanding biological changes ranging from cellular differentiation to epigenetic responses of diseased cells upon drugging. We integrate experiments and theory to determine the trajectories that single BRAFV600E mutant melanoma cancer cells take between drug-naive and drug-tolerant states. Although single-cell omics tools can yield snapshots of the cell-state landscape, the determination of individual cell trajectories through that space can be confounded by stochastic cell-state switching. We assayed for a panel of signaling, phenotypic, and metabolic regulators at points across 5 days of drug treatment to uncover a cell-state landscape with two paths connecting drug-naive and drug-tolerant states. The trajectory a given cell takes depends upon the drug-naive level of a lineage-restricted transcription factor. Each trajectory exhibits unique druggable susceptibilities, thus updating the paradigm of adaptive resistance development in an isogenic cell population.
Subject(s)
Drug Tolerance , Genomics , Melanoma/drug therapy , Single-Cell Analysis , Cell Line, Tumor , Genes, Reporter , Green Fluorescent Proteins/metabolism , Humans , Metabolomics , Microphthalmia-Associated Transcription Factor , Models, Molecular , Proteomics , Proto-Oncogene Proteins B-raf/genetics , Reproducibility of ResultsABSTRACT
Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen-receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG-12RSS-23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG-RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.
Subject(s)
Nucleic Acid Conformation , Protein Sorting Signals/genetics , Receptors, Antigen/genetics , V(D)J Recombination/genetics , Animals , DNA Cleavage , Lymphocytes/metabolism , Single Molecule Imaging , Vertebrates/genetics , Vertebrates/growth & developmentABSTRACT
At the heart of an innate immune response lies a tightly regulated gene expression program. This precise regulation is crucial because small changes can shift the balance from protective to destructive immunity. Here we identify a frequently used alternative splice site in the gene oligoadenylate synthetase 1g (Oas1g), a key component of the 2-5A antiviral system. Usage of this splice site leads to the generation of a transcript subject to decay, and removal of the site leads to increased expression of Oas1g and an improved antiviral response. However, removal of the splice site also leads to an increase in apoptotic cell death, suggesting this splicing event exists as a compromise between the pathogen protective benefits and collateral damage associated with OAS1g activity. Across the innate immune response, we show that a multitude of alternative splicing events predicted to lead to decay exist, and thus have the potential to play a significant role in the regulation of gene expression in innate immunity.
Subject(s)
2',5'-Oligoadenylate Synthetase/metabolism , Alternative Splicing , Antiviral Agents/metabolism , Gene Expression Regulation/genetics , Immunity, Innate/genetics , RNA Splice Sites , 2',5'-Oligoadenylate Synthetase/genetics , Animals , Apoptosis , HEK293 Cells , Humans , Mice , Nonsense Mediated mRNA Decay , RAW 264.7 CellsABSTRACT
NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.
Subject(s)
Cytokine Release Syndrome/prevention & control , Inflammation/prevention & control , Leukemia, Myeloid, Acute/prevention & control , MicroRNAs/genetics , Myeloid Progenitor Cells/pathology , NF-kappa B/metabolism , Animals , Apoptosis , Cell Proliferation , Cytokine Release Syndrome/genetics , Cytokine Release Syndrome/pathology , Female , Gene Expression Regulation , Humans , Inflammation/genetics , Inflammation/pathology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Myeloid Progenitor Cells/metabolism , NF-kappa B/genetics , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T cells using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100 000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of detecting antigen-specific TCRs relative to bulk analysis and simultaneous capture of two virus antigen-specific TCRs from a population of T cells.
Subject(s)
Antigens/genetics , Microfluidic Analytical Techniques , Receptors, Antigen, T-Cell/genetics , CD8-Positive T-Lymphocytes , Cells, Cultured , Humans , Magnetite Nanoparticles/chemistry , Microfluidic Analytical Techniques/instrumentation , Particle Size , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.
Subject(s)
Antigens, Neoplasm/blood , Melanoma/blood , Melanoma/immunology , T-Lymphocytes/immunology , Biopsy , HEK293 Cells , Humans , Immunotherapy , Jurkat Cells , Kinetics , Lymphocytes, Tumor-Infiltrating/immunology , Magnetite Nanoparticles/chemistry , Major Histocompatibility Complex , Melanoma/pathology , Melanoma/secondary , Nucleic Acids/metabolism , Programmed Cell Death 1 Receptor/immunology , Receptors, Antigen, T-Cell/metabolism , Reproducibility of Results , Tomography, X-Ray ComputedABSTRACT
Immunotherapies are yielding effective treatments for several previously untreatable cancers. Still, the identification of suitable antigens specific to the tumour that can be targets for cancer vaccines and T cell therapies is a challenge. Alternative processing of mRNA, a phenomenon that has been shown to alter the proteomic diversity of many cancers, may offer the potential of a broadened target space. Here, we discuss the promise of analysing mRNA processing events in cancer cells, with an emphasis on mRNA splicing, for the identification of potential new targets for cancer immunotherapy. Further, we highlight the challenges that must be overcome for this new avenue to have clinical applicability.
Subject(s)
Alternative Splicing , Immunotherapy/methods , Neoplasms/therapy , Antigen Presentation , Humans , Neoplasms/genetics , Neoplasms/immunology , Receptors, Antigen, T-Cell/immunologyABSTRACT
Tet methylcytosine dioxygenase 2 (Tet2) is an epigenetic regulator that removes methyl groups from deoxycytosine residues in DNA. Tet2-deficient murine macrophages show increased lipopolysaccharide (LPS)-induced and spontaneous inflammation at least partially because Tet2 acts to restrain interleukin (IL)-1ß and IL-6 expression in induced cells. MicroRNAs have emerged as critical regulatory noncoding RNAs that tune immune cell responses to physiological perturbations and play roles in pathological conditions in macrophages. To determine if a microRNA played any role in Tet2 activity, we examined the interrelationship of Tet2 action and the let-7 microRNA family, utilizing several let-7 microRNA engineered murine models. We first showed that Tet2, but not Tet3, is a direct target of the let-7a-1/let-7d/let-7f-1 (let-7adf) microRNAs in macrophages. We found that overexpression or deletion of the let-7adf gene cluster causes altered IL-6 induction both in tissue culture cells induced by LPS treatment in vitro as well as in a Salmonella infection mouse model in vivo. Mechanistically, let-7adf promotes IL-6 by directly repressing Tet2 levels and indirectly by enhancing a Tet2 suppressor, the key TCA cycle metabolite, succinate. We found that Let-7adf promotes succinate accumulation by regulating the Lin28a/Sdha axis. We thereby identify two pathways of let-7 control of Tet2 and, in turn, of the key inflammatory cytokine, IL-6, thus characterizing a regulatory pathway in which a microRNA acts as a feedback inhibitor of inflammatory processes.
Subject(s)
DNA-Binding Proteins/metabolism , Macrophages/metabolism , MicroRNAs/physiology , Protein Processing, Post-Translational , Proto-Oncogene Proteins/metabolism , Animals , Dioxygenases , Interleukin-6/biosynthesis , Interleukin-6/genetics , Lipopolysaccharides , Macrophages/drug effects , Macrophages/enzymology , Mice , Mice, Knockout , RNA, Messenger/genetics , Succinate Dehydrogenase/metabolism , Succinates/metabolismABSTRACT
Immunotherapy treatments harnessing the patient's immune system herald a new era of personalized medicine, offering hope for curative responses in patients with serious illnesses. Cell-mediated immunity can eliminate cancer cells and provide durable remissions. This often relies on repurposing cytotoxic T cell activity through modified T cell receptors or chimeric antigen receptors. Furthermore, synthetic biology has expanded the cell engineering toolkit to provide immune cells with more functionality, including disease targeting, potency, and safety. We focus on T cell-based immunotherapy, highlighting discoveries of genetic engineering and therapeutic use. We also examine emerging paths that could be undertaken to improve this novel class of drugs, and discuss the challenges of toxicities as well as other limitations of cellular immunotherapy.
Subject(s)
Immunotherapy , Neoplasms/therapy , Precision Medicine , Animals , Humans , Neoplasms/immunologyABSTRACT
CD8+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery.
Subject(s)
Antigen Presentation , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , Antigen-Presenting Cells/cytology , Antigens/chemistry , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , CD8-Positive T-Lymphocytes/cytology , Cloning, Molecular , Coculture Techniques , Epitopes/chemistry , False Positive Reactions , Gene Library , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Immunotherapy/methods , Jurkat Cells , K562 Cells , Lectins, C-Type/metabolism , Major Histocompatibility Complex , Oligonucleotides/genetics , Peptides/chemistryABSTRACT
T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide-major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide-MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy.
Subject(s)
Antigens/chemistry , Genetic Techniques , Receptors, Antigen, T-Cell/chemistry , T-Lymphocytes/cytology , Adaptive Immunity , Animals , Biotinylation , DNA/analysis , Epitopes/chemistry , Gene Library , HEK293 Cells , Humans , Immunotherapy , Jurkat Cells , K562 Cells , Ligands , Mice , Peptides/chemistry , Phagocytosis , T-Lymphocytes/immunologyABSTRACT
Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs.
Subject(s)
Interferon Regulatory Factor-7/metabolism , Interferon Type I/metabolism , Introns , Macrophages/metabolism , RNA-Binding Proteins/metabolism , Vesicular Stomatitis/metabolism , Vesicular stomatitis Indiana virus/pathogenicity , Animals , Binding Sites , Chlorocebus aethiops , GC Rich Sequence , HEK293 Cells , Host-Pathogen Interactions , Humans , Interferon Regulatory Factor-7/genetics , Interferon Type I/immunology , Macrophages/immunology , Macrophages/virology , Mice, Inbred C57BL , Protein Binding , RNA Splice Sites , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Vero Cells , Vesicular Stomatitis/genetics , Vesicular Stomatitis/immunology , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/immunologyABSTRACT
Each of us is a story. Mine is a story of doing science for 60 years, and I am honored to be asked to tell it. Even though this autobiography was written for the Annual Review of Immunology, I have chosen to describe my whole career in science because the segment that was immunology is so intertwined with all else I was doing. This article is an elongation and modification of a talk I gave at my 80th birthday celebration at Caltech on March 23, 2018.
Subject(s)
Allergy and Immunology/history , NF-kappa B/metabolism , RNA Viruses/physiology , Virus Diseases/immunology , Animals , Disease Models, Animal , Gene Rearrangement , History, 20th Century , History, 21st Century , Humans , Mice , Protein-Tyrosine Kinases/metabolism , Reverse Transcription , United StatesABSTRACT
PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.
Subject(s)
Genetic Therapy/methods , Hematopoietic Stem Cells/immunology , Immunotherapy, Adoptive/methods , Neoplasms/therapy , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Animals , Antigens, Neoplasm/genetics , Cells, Cultured , Clinical Trials as Topic , Drugs, Investigational/therapeutic use , HLA-A2 Antigen/genetics , Hematopoietic Stem Cells/metabolism , Humans , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms/genetics , Neoplasms/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolismABSTRACT
Long-term hematopoietic stem cells (LT-HSCs) maintain hematopoietic output throughout an animal's lifespan. However, with age, the balance is disrupted, and LT-HSCs produce a myeloid-biased output, resulting in poor immune responses to infectious challenge and the development of myeloid leukemias. Here, we show that young and aged LT-HSCs respond differently to inflammatory stress, such that aged LT-HSCs produce a cell-intrinsic, myeloid-biased expression program. Using single-cell RNA sequencing (scRNA-seq), we identify a myeloid-biased subset within the LT-HSC population (mLT-HSCs) that is prevalent among aged LT-HSCs. We identify CD61 as a marker of mLT-HSCs and show that CD61-high LT-HSCs are uniquely primed to respond to acute inflammatory challenge. We predict that several transcription factors regulate the mLT-HSCs gene program and show that Klf5, Ikzf1, and Stat3 play an important role in age-related inflammatory myeloid bias. We have therefore identified and isolated an LT-HSC subset that regulates myeloid versus lymphoid balance under inflammatory challenge and with age.
Subject(s)
Aging/pathology , Hematopoietic Stem Cells/metabolism , Inflammation/pathology , Animals , Biomarkers/metabolism , Inflammation/genetics , Ligands , Mice, Inbred C57BL , Models, Biological , Myeloid Cells/metabolism , Toll-Like Receptors/metabolism , Transcription, GeneticABSTRACT
Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.